Proteolysis by the ubiquitin system plays major roles in a multitude of biological processes. Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. This is the resubmission of renewal application for the DK39520 grant, currently in its 25th year of support. Two of several discoveries during the preceding period of support are the new functions of the N-end rule pathway as a sensor, through different mechanisms, of both heme and nitric oxide. Another discovery is the identification and analysis of an entirely new class of aminoacyl-transferases that underlie the N-end rule pathway in prokaryotes. One focus of the present renewal application is on physiological substrates, mechanisms and functions of ubiquitin ligases in both yeast and mammalian N-end rule pathways. Hence the (overdue) change of DK39520 title, from Mechanics and Functions of the Yeast N-End Rule Pathway to Ubiquitin Ligases, Mechanisms and Functions of the N-End Rule Pathway. This title more accurately describes the scope (including the range of model organisms) of our research supported by the DK39520 grant. Updated Specific Aims: 1) Exploration of the discovery that S. cerevisiae MGT1, a key DNA repair enzyme, is a physiological substrate of two distinct Ub ligases, UBR1 and UFD4. Extension of these insights to MGMT, the mammalian counterpart of yeast MGT1. 2) Development and applications of a new method, termed the N-degron capture (NDC), for discovering physiological substrates of the N-end rule pathway. This method is based on an altered genetic code, a photoactivatable ClpS (the recognition component of the E. coli N-end rule pathway), and target- selective UV-crosslinking in intact cells or in vitro. 3) Studies of S. cerevisiae UBR1 and the mouse UBR-family Ub ligases of the N-end rule pathway, including their crystal structures, further explorations of their recently discovered interactions with heme, as well as their physiological substrates. 4) The deamidation branch of the N-end rule pathway: isolation, cloning and molecular genetic dissection of the mouse NTAQ1, a glutamine-specific N-terminal amidase, NtQ-amidase. The present resubmission application for the renewal of the present grant contains substantial experimental updates (produced during the last 6-7 months) of most Aims. PUBLIC HEALTH RELEVANCE The proposed studies in this renewal application, based in part on our recent discoveries, will illuminate the mechanisms and functions of regulated protein degradation in yeast and mammals, including humans. Either inborn or acquired defects in these systems, which include the ubiquitin system, are a major cause of many human diseases, including cancer and neurodegenerative syndromes. Understanding, in depth and detail, the mechanisms and functions of the ubiquitin system will result in better therapies for these and other medical problems, including currently intractable ones.